The objectives of the proposed research are to understand the enzymology of human methionine synthase (MS) and methionine synthase reductase (MSR) as well as their clinically relevant variants. MSR is a key enzyme of folate and cobalamin metabolism involved in the reductive activation of MS. MS is required for the folate/cobalamin-dependent conversion of homocysteine and cycles between the highly reactive cob(I)alamin and the cob(III)alamin species. Overtime MS becomes inactive when cob(I)alamin is oxidized to the inert cob(II)alamin form. MSR reactivates MS through reductive methylation of cob(II)alamin with S-adenosyl methionine acting as the methyl donor. The NADPH-derived reducing equivalents are transferred through two flavin cofactors (FAD and FMN) located in MSR. MSR and individual flavin-binding domains have been cloned into His-tagged expression vectors for expression in E. coli and purification via Ni-affinity chromatography. The stoichiometry of flavin binding and the flavin redox potentials will be determined for purified MSR and the individual flavin-binding domains. An array of stopped-flow and transient kinetic studies will be performed on MSR and the individual FAD and FMN-binding domains to investigate the following: (1) the rate of formation of charge-transfer complexes between NADPH and FAD; (2) the forward and reverse rate of hydride transfer; (3) the NADPH concentration dependence of hydride transfer, and (4) the rate of intra-flavin electron. The research, which is detailed in this proposal, will investigate the thermodynamic and kinetic mechanism of electron transfer in wild type MSR, to provide a framework for understanding the molecular basis of clinically important variants of the enzyme that contribute to cardiovascular disease and impaired fetal development.